Laboratory of Pharmacology, Department of Drug Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal.
Parasite Disease Group, Institute of Molecular and Cellular Biology, Institute for Research and Innovation in Health Sciences, University of Porto, Rua Alfredo Allen 208, 4200-135, Porto, Portugal; Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal.
Eur J Pharmacol. 2020 Oct 15;885:173504. doi: 10.1016/j.ejphar.2020.173504. Epub 2020 Aug 26.
Leishmania infected macrophages have conditions to produce adenosine. Despite its known immunosuppressive effects, no studies have yet established whether adenosine alter Leishmania parasitic burden upon macrophage infection. This work aimed at investigating whether endogenous adenosine exerts an autocrine modulation of macrophage response towards Leishmania infection, identifying its origin and potential pharmacological targets for visceral leishmaniasis (VL), using THP-1 differentiated macrophages. Adenosine deaminase treatment of infected THP-1 cells reduced the parasitic burden (29.1 ± 2.2%, P < 0.05). Adenosine A and A receptor subtypes expression was confirmed by RT-qPCR and by immunocytochemistry and their blockade with selective adenosine A and A antagonists reduced the parasitic burden [14.5 ± 3.1% (P < 0.05) and 12.3 ± 3.1% (P < 0.05), respectively; and 24.9 ± 2.8% (P < 0.05), by the combination of the two antagonists)], suggesting that adenosine A receptors are tonically activated in infected THP-1 differentiated macrophages. The tonic activation of adenosine A receptors was dependent on the release of intracellular adenosine through equilibrative nucleoside transporters (ENT1/ENT2): NBTI or dipyridamole reduced (~25%) whereas, when ENTs were blocked, adenosine A receptor antagonists failed to reduce and A agonists increase parasitic burden. Effects of adenosine A receptors antagonists and ENT1/2 inhibitor were prevented by L-NAME, indicating that nitric oxide production inhibition prevents adenosine from increasing parasitic burden. Results suggest that intracellular adenosine, released through ENTs, elicits an autocrine increase in parasitic burden in THP-1 macrophages, through adenosine A receptors activation. These observations open the possibility to use well-established ENT inhibitors or adenosine A receptor antagonists as new therapeutic approaches in VL.
利什曼原虫感染的巨噬细胞有产生腺苷的条件。尽管腺苷具有已知的免疫抑制作用，但尚无研究确定腺苷是否会改变巨噬细胞感染利什曼原虫时的寄生虫负担。本研究旨在通过使用分化的 THP-1 巨噬细胞，研究内源性腺苷是否对巨噬细胞对利什曼原虫感染的反应产生自分泌调节作用，确定其来源和潜在的药物靶点，用于内脏利什曼病（VL）。腺苷脱氨酶处理感染的 THP-1 细胞可降低寄生虫负担（29.1±2.2%，P<0.05）。通过 RT-qPCR 和免疫细胞化学证实了腺苷 A 和 A 受体亚型的表达，并通过选择性腺苷 A 和 A 拮抗剂阻断其表达可降低寄生虫负担[14.5±3.1%（P<0.05）和 12.3±3.1%（P<0.05）；两种拮抗剂联合使用时，寄生虫负担降低 24.9±2.8%（P<0.05）]，表明腺苷 A 受体在感染的 THP-1 分化巨噬细胞中持续激活。腺苷 A 受体的持续激活依赖于通过平衡核苷转运蛋白（ENT1/ENT2）从细胞内释放腺苷：NBTI 或双嘧达莫可降低（约 25%）；而当 ENTs 被阻断时，腺苷 A 受体拮抗剂不能降低寄生虫负担，而腺苷 A 激动剂则增加寄生虫负担。腺苷 A 受体拮抗剂和 ENT1/2 抑制剂的作用被 L-NAME 阻止，表明一氧化氮产生抑制可防止腺苷增加寄生虫负担。结果表明，通过 ENTs 释放的细胞内腺苷通过激活腺苷 A 受体引发 THP-1 巨噬细胞中寄生虫负担的自分泌增加。这些观察结果为使用成熟的 ENT 抑制剂或腺苷 A 受体拮抗剂作为 VL 的新治疗方法提供了可能性。
